Csir - Ugc (Net) : Life Sciences
Csir - Ugc (Net) : Life Sciences
Csir - Ugc (Net) : Life Sciences
Life Sciences
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Developmental Biology
Potency is the capacity to differentiate into specialized cell types. In the strictest sense, this
requires stem cells to be either totipotent or pluripotent – to be able to give rise to any mature cell type,
although multipotent or unipotent progenitor cells are sometimes referred to as stem cells.
1. Totipotent Stem cells are produced from the fusion of an egg and sperm cell. Cells
produced by the first few divisions of the fertilized egg are also totipotent. These cells can
differentiate into embryonic and extraembryonic cell types.
2. Pluripotent Stem cells are the descendants of totipotent cells and can differentiate into
cells derived from any of the three germ layers.
Fig. : Stages of Cell Commitment : Specification (Reversible) and Determination (Essentially Irreversible)
Specification Types
(a) Autonomous specification
(b) Syncytial specification
(c) Conditional specification
Autonomous Specification
Cells are specified by differential distribution of cytoplasmic components during cleavage of the
egg and early embryo.
Modes of Cell Type Specification and Their Characteristics
Characteristics of most invertebrates.
Specification by differential acquisition of certain cytoplasmic molecules present in the
egg.
Invariant cleavages produce the same lineages in each embryo of the species. Blastomere
fates are generally invariant.
Cell type specification precedes any large-scale embryonic cell migration.
Produces ‘mosaic’ development cells cannot change fate if a blastomere is lost.
Fig. : Autonomous specification in Tunicate (sea squirt). Blastomeres are committed at a very early
stage in mosaic development. If split, each dissociated blastomere pair forms original structures.
Each blastomere contains positional information in the form of specific proteins and genes.
Syncytial Specification
Nuclear division without cell division, results in cytoplasm with many nuclei.
Fig. : Ectodermal Ability to Respond to The Optic Vesicle Inducer in Xenopus Laevis
1. The optic vesicle is able to induce lenses in the anterior portion of the ectoderm, but not
in the presumptive trunk and abdomen.
2. If the optic vesicle is removed.
3. The surface ectoderm forms either an abnormal lens or no lens at all.
4. Most other tissues are not able to substitute for the optic vesicle.
Competence is ability to respond to a specific inductive signal. Competence is an actively acquired
condition.
Fig. : Inductive Interactions : Interaction between Epithelia and Mesenchyme : Mesenchyme Plays
An Instructive Role (as The Inducing Tissue); and Initiates Gene Activity in Epithelial Cells.
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Fig. : Pax6 is a competence factor for lens induction. During lens induction Pax6 expressed
in the head ectoderm, but not in other regions of surface ectoderm.
Mesenchymal-Epithelial Interactions
Many inductions involve interactions between epithelia and mesenchyme. Mesenchyme initiates
gene activity in epithelial cells.
Fig. : Epithelial derivatives (hair, scales, feathers, mammary glands, sweat glands)
type depends on restrictions by region and genetics.
In regional specificity, source of the mesenchyme (inducing tissue) determines the structure
of the epithelial derivative.
(a) The bicoid mRNA (messenger RNA) is an intermediate copy of a piece of the DNA. It is
transported to the ribosomes, where protein biosynthesis takes place) is located on the
left side of the embryo. All cells of the embryo have inactive hunchback DNA.
(b) The bicoid mRNA is expressed as bicoid protein, resulting in a protein gradient with the
highest concentration of protein on the left side of the embryo.
(c) The hunchback DNA is activated once the amount of bicoid protein passes a certain
threshold. This results in a sharp borderline, which divides the part, where hunchback is
expressed from the part where hunchback is not expressed.
Dorso-ventral (front-and backside)
Terminal (special structures at the unsegmented ends of the embryo)
In the later stages of development, these basic compartments become more and more divided.
The same transcription factor can be used several times, having different meanings in the different body
parts.
The term ‘morphogenesis’ can be used to describe the development of unicellular life forms that
do not have an embryonic stage in their life cycle. Morphogenetic responses may be induced in organisms
by hormones or by environmental chemicals.
Cell Fate and Cell Lineages
Fate is the sum of all structures that the cell or its descendants will form at a later stage of normal
development.
It is the ultimate differentiated state to which a cell has become committed. Fate maps are the
bases for experimental embryology
The cell lineage of an organism is the pattern of cell divisions during its development. Cell
lineages are described by following cell divisions in living individuals or by marking cells and examining
their progeny.
Cell Lineage Patterns
(i) Some organisms or precursor cells display cell lineage patterns are invariant patterns of
cell division, in which specification of cell fates is correlated with cell division patterns.
(ii) In other organisms, cell lineage patterns are variable and not correlated with cell fates.
Invariant cell lineages reflect both cell-autonomous mechanisms of fate determination and
highly reproducible cell-cell interactions. Genetic analysis of cell lineages has focused on
systems, where cell lineage and cell fates are correlated.
Fate Mapping Methods
Fate maps have been generated in several ways:
(i) Observing living embryos In certain invertebrates, it is actually possible to look through
the microscope and trace the descendants of a particular cell into the organs they generate.
This type of study was performed about a century ago by Edwin G Conklin.
Fig. : Vital dye staining of amphibian embryos. (a) Specific cells of the embryonic surface with vital dyes.
(b-d) Dorsal surface views of stain on successively later embryos. (e) Medial sagittal section to
show the stained cells
(iii) Radioactive labelling and fluorescent dyes In this method, a donor embryo is usually
grown in a solution containing radioactive thymidine. This base will be incorporated into
the DNA of the dividing embryo.
The problems with radioactive and vital dye marking include their dilution over many cell divisions
and the laborious preparation of the slides.
Methods of Analyses of Cell Lineage
(i) Direct observation Same as described above under ‘Fate Mapping Methods’.
(ii) Clonal analysis In large, opaque or slowly developing embryos, direct observation of cell
divisions is not feasible. To analyze cell lineages in such cases, it is necessary to mark
individual cells by physical or genetic means and later to identify their progeny by expression
of the marker. Here, the progeny of a single cell forms a clone.
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Stem Cells
Stem cells are a class of undifferentiated cells (i.e., lacking certain tissue specific differentiation
markers) that are able to differentiate into specialized cell types.
All stem cells have three general properties.
1. Stem cells are capable of dividing and renewing themselves for long periods.
2. Stem cells are unspecialized.
3. Stem cells can give rise to specialized cells.
The two broad types of mammalian stem cells are embryonic stem cells that are found
in blastocysts and adult stem cells that are found in adult tissues.
In a developing embryo, stem cells can differentiate into all of the specialized embryonic
tissues.
4. Embryonic stem cells are primitive (undifferentiated) cells from the embryo that have the
potential to become a wide variety of specialized cell types.
5. Human embryonic stem cells are derived from blastocyst.
Embryonic stem cells develop from eggs that have been fertilized invitro or invivo. Another
potential source of embryonic stem cells is Somatic Cell Nuclear Transfer (SCNT). Adult
stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ.
One population is haematopoietic stem cells and the second population is bone marrow
stromal cells.
Genomic Equivalence and The Cytoplasmic Determinants
Genomic equivalence means that the genornes of all or most of the somatic cells within a
multicellular organism are essentially the same. Thus, the differentiation of cells does not (usually)
involve changes in the nucleotide sequence of DNA. Genomic equivalence can be demonstrated via
various cloning experiments including the growing of a whole plant from an isolated cell (other than a
zygote) and the cloning of an animal via the injection of a somatic-cell genome into a zygot (or unfertilized
egg) whose own genome has been destroyed.
Genomic Equivalence for Plants and Animals
Genomic equivalence; nearly all of the cells of an organism have the same genes.
Because the cells of animals will not often divide in culture, scientists have adopted
alternative approaches to examine genomic equivalence: transplanting nuclei of differentiated
cells into enucleated egg cells of frogs.
Plants’ genomic equivalence was demonstrated by experiments in which entire individuals
developed from differentiated somatic cells.
Fig. : Procedure for transplanting blastula nuclei into activated enucleated Rana pipiens eggs.
The relative dimensions of the meiotic spindle have been exaggerated to show the technique
Fig. : Nuclei of Adult Somatic Cells are used for Cloning Mammals
(a) The upper left dark mouse is the oocyte donor, while the upper right light (agouti) mouse
is the nucleus donor. The white mouse in the lower row is the mouse into whose uterus
the resulting embryos were implanted. The two agouti mice beside her are cloned mice,
derived from the injection of the agouti nucleus into the oocyte of the black-furred parent.
(b) Similarly, Dolly, the adult sheep, was derived by fusing a mammary gland cell nucleus with
an enucleated oocyte, which was then implanted in a surrogate mother (of a different
breed of sheep who gave birth to dolly.)
Table : Examples of Genomic Non-equivalence During Development
Mechanism Examples
DNA amplification Selective amplificaton of rRNA genes in Xenopus oocytes.
Amplification of the chorion genes during oogenesis in
Drosophila.
DNA loss In mammals, loss of nuclei in differentiated erythrocytes
and keratinocytes.
DNA rearrangement In vertebrates, VDJ recombination and class switching
during B-cell and T-cell development.
A cytoplasmic determinant is a substance or substances, located in part of an egg or blastomere,
that guarantees the assumption of a particular state of commitment by the cells which inherit it during
cleavage.
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Cytoplasmic determinants are of considerable importance for the very earliest stages of embryonic
development because they are often responsible for the establishment of the first two or three distinctly
specified regions in the embryo. There are established experimental techniques that reorganize the
cytoplasm and thus redistribute the determinants or block their transport. For example, the transfer of
cytoplasm from the anterior pole to the posterior of the Drosophila egg by microinjection demonstrates
the role of polarized maternal cytoplasmic determinants in axis specification, since the embryo develops
with two heads and no abdomen.
In the early stages of C. elegans development a complex containing PAR3, PAR6 and aPKC
becomes localized in the anterior and controls the fate of the first two blastomeres. A similar system
seems to be involved in later events of neurogenesis and epithelial polarization in other animals.
Imprinting
Genomic imprinting is an epigenetic phenomenon which, in most cases, is believed to occur in
gametogenesis. Genomic imprinting occurs when both maternal and paternal alleles are present, but one
allele will be expressed, while the other remains inactive. It is not completely evident why genes are
imprinted. The most prominent assumption is that this process is necessary for development and may
somehow regulate growth in the embryo and neonate. Evidence for this suggestion came from experiments
with androgenotes (embryos with two paternal genomes) and gynogenotes (embryos with two maternal
genomes), which were produced by nuclear transplantation. These zygotes were formed, but neither
type was able to undergo further development. From this situation, it is possible to suggest that the
maternal and paternal effects are complementary.
The optimal method for gene imprinting is DNA methylation. This process is carried out with the
enzyme DNA methyltransferase (DNA MTase) in mammals. DNA MTase acts on the DNA sequence 5-
CpG-3. Some organisms (primarily higher eukaryotes) have aggregates of CpG (known as CpG islands)
in their genomes. These islands are rarely methylated in animal cells. This may be due to the bound
transcription factors that block DNA MTase.
Mutants and Transgenics in Analysis of Development
Recently developed techniques have enabled us to study gene function by moving certain genes
into and out of embryonic cells.
Technique of Inserting DNA into a Cell
Cloned pieces of DNA can be isolated, modified (if so desired) and inserted into cells by several
means. Microinjection, in which a solution containing the cloned gene is injected into the nucleus of a
cell. This technique is especially useful for inserting genes into newly fertilized eggs, since the eggs are
relatively large. Transfection, DNA fragments may be incorporated directly into cells by incubating the
cells in a solution containing new DNA. The chances of a DNA fragment incorporation into the chromosomes
are relatively small. Electroporation, in which a high-voltage pulse ‘pushes’ the DNA into the cells.
Transposable element or retroviral vector, a more ‘natural’ way of getting genes into cells is to insert
the cloned gene into a transposable element or retroviral vector. These naturally occurring mobile regions
of DNA can integrate themselves into the genome of an organism.
Chimeric Mice Experiments
In the blastocyst, there is a stage when two cell types are present: The outer trophoblast cells,
which will form the fetal portion of the placenta and the Inner Cell Mass (ICM), whose cells will give rise
to the embryo itself. Separation of ICM cells can lead to twins and if an 1CM blastomere from one mouse
is transferred into the embryo of a second mouse, the donor 1CM cell can contribute to every organ of
the host embryo.
Inner cell mass blastomeres can be isolated from an embryo and cultured in vitro, such cultured
cells are called embryonic stem cells (ES cells). ES cells are almost totipotent, except the trophoblast.
Embryonic stem cells from a mouse are cultured and their genome altered by the addition of a
cloned gene. These transgenic cells are selected and then injected into the early stages of a host mouse
embryo.
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The transgenic embryonic stem cells integrate with the host’s embryonic stem cells. The embryo
is placed into the uterus of a pregnant mouse, where it develops into a chimeric mouse. The treated
cells can become part of the germ line of the mouse. If such a chimeric mouse is mated with a wild-
type mouse, some of its progeny will carry one copy of the inserted gene. When these heterozygous
progeny are mated to one another, about 25 percent of the resulting offspring will be homozygous for
the inserted gene in every cell of their bodies. Thus, a gene cloned from some other organism will be
present in both copies of the chromosomes within these mouse genomes. Strains of such transgenic
mice have been particularly useful in determining how genes are regulated during development.
To find the function of Bmp7 in the development of the mouse. First, isolate the (Bmp7) gene,
cut it at one site with a restriction enzyme and inserted a bacterial gene for neomycin resistance into
that site. This mutated the Bmp7 gene has destroyed the ability of the gene’s product (Bmp7 protein)
to function. These mutant (Bmp7) genes were electroporated into embryonic stem cells that were
naturally sensitive to neomycin. Once inside the nucleus of an ES cell, the mutated Bmp7 gene may
replace a normal allele of Bmp7 by a process called homologous recombination. Thus, the enzymes
involved in DNA repair and replication incorporate the mutant gene in the place of the normal copy. It’s
a rare event, but such cells can be selected by growing the ES cells in neomycin. The resulting cells
have one normal Bmp7 gene and one mutated Bmp7 gene. These heterozygous ES cells were then
microinjected into mouse blastocysts, where they were integrated into the cells of the embryo. The
resulting chimeric mice had wild-type cells from the host embryo and heterozygous Bmp 7-containing
cells from the donor ES cells. The chimeras were mated to wild-type mice, producing progeny that were
heterozygous for Bmp7. These heterozygous mice were then bred with each other and about 25 percent
of their progeny carried two copies of the mutated Bmp7 gene.
The homozygous mutants lacked eyes and kidneys. In the absence of functional Bmp7 protein,
it appears that many of the cells that normally form these two organs stop dividing and die. In this way,
gene targeting can be used to analyze the roles of particular genes during mammalian development.
Fig. : Technique for Gene Targeting. In this case, the targeted gene is Bmp7
(a) Embryonic stem cells from a mouse blastocyst are cultured.
(b) Cloned Bmp7 genes are cut with a restriction enzyme and a neomycin resistance gene
is inserted into the region that encodes the DNA-binding site of the protein. These mutant
Bmp7 genes are electroporated into ES cells. In some of the cells, homologous
recombination exchanges a wild-type gene for the mutant copy. These cells are selected
by their neomycin resistance.
1. In higher plants, the red/far-red sensory photoreceptor, phytochrome, is a light regulated kinase.
Which of the following classes of kinase does it represent ?
(A) Two-component sensor regulator (histidine kinase)
(B) Two-component sensor regulator (serine/threonine kinase)
(C) Reprogramming of the shoot apical meristem
(D) Synthesis of the flowering hormone florigen
7. A Graffian follicle is
(A) An immature developing follicle (B) A mature follicle ready to ovulate
(C) A follicle undergoing apoptosis (D) An ovulated follicle
8. The diagram represents the top view of a developing egg at 8 celled stage of cleavage. Which
one of the following group of animals show this pattern
9. An embryo lacking bicoid is injected with bicoid m-RNA at middle portion. It will result into
(A) Two heads and no telson
(B) Head in the middle and telson at both ends
(C) No head and telson at both ends
(D) Normal phenotype
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